The innate immune response to an invading pathogen involves the effective and rapid recognition of highly conserved and repeated foreign structures such as those found in polysaccharides, lectins, complexed lipids (e.g., LPS) and double stranded (ds) RNA. Medzhitov and Janeway (1997) Cell 91:295. Recently, bacterial genomic DNA, plasmids and immunostimulatory oligodeoxynucleotides containing CpG dinucleotides in a particular base context (ISS-ODN or CpG motifs) have been shown to activate innate immunity. Klinman et al. (1999) Immunity 11:123. In contrast, mammalian DNA or methylated bacterial DNAs are inactive. ISS stimulate macrophages/monocytes to secrete IL-6 and IL-12, activate NK cells, induce B-cell proliferation and polyclonal IgM production and rescue B cells from apoptosis. Klinman et al. (1999) Immunity 11:123. Furthermore, when ISS is co-delivered with an antigen, it elicits cell-mediated immunity, which mimics the host immune response against viral infection. Roman et al. (1997) Nature Med. 8:849.
Ku protein was originally discovered as an autoantigen recognized by autoantibodies from the sera of certain patients with systemic autoimmune diseases. Mimori et al. (1981) J. Clin. Invest. 68:611-620; and Mimori and Hardin (1986) J. Biol. Chem. 261:10375-10379; and reviewed in Reeves et al. (1992) Rhem. Dis. Clin. North Am. 18:391-414. Ku antigen is a heterodimeric protein consisting of two polypeptides of approximately 70 kDa and 80 kDa. Ku antigen was subsequently shown to the regulatory component of DNA-dependent protein kinase (DNA-PK). Dynan and Yoo (1998) Nucl. Acids Res. 26:1551-1559.
Ku, which binds to double-stranded DNA breaks (DSB), is believed to play a role in targeting the DNA-PK complex to DSB for repair. Specifically, when bound to DNA, Ku interacts with and activates the DNA-PK catalytic subunit (DNA-PKcs). DNA-PKcs is believed to interact with and phosphorylate several DNA-binding proteins in vitro, such as replication protein A and the tumor suppressor protein p53, respectively, as well as other transcription factors. Anderson and Lees-Miller (1992) Crit. Reviews in Euk. Gene Expression 2:283; and Anderson (1993) Trends Biochem. Sci. 18:433. DNA-PK is thought to play a role in controlling gene regulation and cell growth. Ku has been reported to bind to various DNA sequences, including NRE1 (negative regulatory element 1) sequences from a viral LTR comprising repeats of 5′-GAAAG-3′ (Giffin et al. (1997) J. Biol. Chem. 272:5647-5658); an Alu core element comprising the sequence 5′-GGAGGGC-3′ (Tsuchiya et al. (1998) J. Biochem. 123:120-127; and a mammalian DNA origin of replication (Ruiz et al. (1999) Mol. Biol. Cell 10:567-580).
In addition to DSB repair, DNA-PK is also involved in V(D)J recombination, isotype switching, as well as telomere length maintenance and silencing. Weaver et al. (1996) CRC Crit. Rev. Eukaryotic Gene Exp. 6:345-375; Chu (1997) J. Biol. Chem. 272:24097-24100; Casellas et al. (1998) EMBO J. 17:2404-2411; and Boulton and Jackson (1998) EMBO J. 17:1819-1828. DNA-PK also participates in the activation of NFκB by ioninizing radiation. Basu et al. (1998) Biochem. Biophys. Res. Comm. 247:79-83.
Apoptosis, or programmed cell death (PCD) is a type of cell death that is fundamentally distinct from degenerative death or necrosis. It is an active process of gene-directed cellular self-destruction which in some instances, serves a biologically meaningful homeostatic function.
Apoptotic cell death is characterized primarily by internucleosomal DNA cleavage and chromatin condensation, and also by cellular shrinkage, cytoplasmic blebbing, and increased membrane permeability. Gerschenson et al. (1992) FASEB J. 6:2450-2455; and Cohen and Duke (1992) Ann. Rev. Immunol. 10:267-293. This can be contrasted to necrosis, which is cell death occurring as the result of severe injurious changes in the environment of infected cells. Necrosis is characterized by the swelling and rupturing of cells, the loss of membrane integrity, a random breakdown of DNA into fragments of variable size, and the phagocytosis of cellular debris by macrophages. The release of lysosomal enzymes damages neighboring cells, thus, cells die in groups. This produces an inflammatory response in tissue. Cell death by necrosis involves no direct RNA or protein synthesis. For a general review of apoptosis, see Tomei, L. D. and Cope, F. O. Apoptosis: The Molecular Basis of Cell Death (1991) Cold Spring Harbor Press, N.Y.; Tomei, L. D. and Cope, F. O. Apoptosis II: The Molecular Basis of Apoptosis in Disease (1994) Cold Spring Harbor Press, N.Y.; and Duvall and Wyllie (1986) Immun. Today 7:115-119.
Apoptosis can be activated by a number of intrinsic or extrinsic signals. These signals include the following: mild physical signals, such as ionization radiation, ultraviolet radiation, or hyperthermia; low to medium doses of toxic compounds, such as azides or hydrogen peroxides; chemotherapeutic drugs, such as etoposides and teniposides, cytokines such as tumour necrosis factors and transforming growth factors; infection with human immunodeficiency virus (HIV); and stimulation of T-cell receptors. Various pathological processes, such as hormone deprivation, growth factor deprivation, thermal stress and metabolic stress, induce apoptosis. (Wyllie, A. H., in Bowen and Lockshin (eds.) Cell Death in Biology and Pathology (Chapman and Hall, 1981), at 9-34).
Unregulated apoptosis can cause, or be associated with, disease. For example, unregulated apoptosis is involved in diseases such as cancer, heart disease, neurodegenerative disorders, autoimmune disorders, and viral and bacterial infections. Cancer, for example, not only triggers cells to proliferate but also blocks apoptosis. Cancer is partly a failure of apoptosis in the sense that the signal(s) for the cells to kill themselves by apoptosis are blocked.
In heart disease, damage caused by trauma (e.g, resulting in shock), and cardiac cells can be induced to undergo apoptosis. For example, cells deprived of oxygen after a heart attack release signals that induce apoptosis in cells in the heart. Apoptosis may also be involved in the destruction of neurons in people afflicted by strokes or neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS). There is also evidence suggesting that ischemia can kill neurons by inducing apoptosis. It has been shown that neurons that are resistant to apoptosis are also resistant to ischemic damage, thus, inhibition of apoptosis may be a therapeutic strategy for the treatment of neurodegenerative or cardiovascular disorders, e.g., stroke.
Under normal physiological conditions, self-reactive immune cells may be induced to undergo apoptosis, thereby removing such self-reactive cells. A failure of the immune system to induce apoptosis in a self-reactive immune cell can lead to autoimmune disease. For example, autoimmune diseases such as rheumatoid arthritis, diabetes, and multiple sclerosis, result when a small percentage of T cells attack the body's own tissue.
There is a need in the art for methods of modulating cell death resulting from genotoxic insults. The present invention addresses this need, and provides related advantages as well.